1. Field of the Invention
The present invention concerns a process for rendering a biological composition substantially free of enveloped and non-enveloped viruses contained therein without substantial disruption or inactivation of cells contained therein and without significant loss of labile proteins or other valuable biological components also contained therein.
2. Description of Related Art
The problems associated with the application of virucidal procedures to biological compositions and the efforts to date to overcome these problems, including the application of light and chemical agents is reviewed briefly in U.S. Pat. No. 5,120,649, the disclosure of which is incorporated herein by reference. See column 1, line 27, through column 4, line 41, therein.
Various photodynamic sterilization techniques have been evaluated for inactivating viruses in cellular components of blood. Although many of these appear promising for the treatment of red cell concentrates (Matthews et al., xe2x80x9cPhotodynamic therapy of viral contaminants with potential for blood banking applicationsxe2x80x9d, in Transfusion, 28:81-83 (1988); O""Brien et al., xe2x80x9cEvaluation of merocyanine 540-sensitized photoirradiation as a means to inactivate enveloped viruses in blood productsxe2x80x9d, in J. Lab. Clin. Med., 116:439-47 (1990); and Horowitz et al., xe2x80x9cInactivation of viruses in blood with aluminum phthalocyanine derivativesxe2x80x9d, in Transfusion, 31:102-8 (1991)), photodynamic viral inactivation methods involving solely oxygen dependent reactions have so far proved inappropriate for the treatment of platelet concentrates (Proudouz et al., xe2x80x9cInhibition by albumin of merocyanine 540-mediated photosensitization of platelets and virusesxe2x80x9d, in Transfusion, 31:415-22 (1991), Dodd et al., xe2x80x9cInactivation of viruses in platelet suspensions that retain their in vitro characteristics: comparison of psoralen-ultraviolet A and merocyanine 540-visible light methodsxe2x80x9d, in Transfusion, 31:483-90 (1991); and Horowitz et al., xe2x80x9cInactivation of viruses in red cell and platelet concentrates with aluminum phthalocyanine (AIPc) sulfonatesxe2x80x9d, in Blood Cells, 18:141-50 (1992)).
One of the latest developments is the use of photoactive compounds. See, e.g., U.S. Pat. No. 5,120,649 and U.S. Ser. No. 07/706,919, filed May 29, 1991. Psoralen, together with UVA, has been shown to kill viruses in both cell-containing and cell-free solutions without undue damage to the valuable components needed for transfusion. Methylene blue, together with visible light, is being used to treat whole plasma. Phthalocyanines and other heme analogs, together with visible light, are being explored for treatment of red blood cell concentrates and other blood components.
Treatment with psoralens and long wavelength ultraviolet light (UVA) is known to produce various biochemical effects including oxygen independent interactions with nucleic acids (e.g., psoralen-DNA monoadduct formation and DNA crosslinking) and oxygen dependent reactions of a photodynamic nature (for review, see Gasparro, F. P. (Ed.) (1988) Psoralen DNA Photobiology, Vol I, Vol II, CRC Press, Boca Roton, Fla.). In contrast to the purely photodynamic procedures appropriate for red cells (above), the use of psoralens and UVA has demonstrated promise as a means of photoinactivating viral contaminants in platelet concentrates, although in most studies (Lin et al., xe2x80x9cUse of 8-methoxypsoralen and long-wavelength ultraviolet radiation for decontamination of platelet concentratesxe2x80x9d, in Blood, 74:517-525 (1989); and Dodd et al., supra, aminomethyl-trimethylpsoralen (AMT)), the combination of high levels of virus inactivation and the maintenance of platelet function were possible only when air was exchanged with nitrogen prior to UVA irradiation, a cumbersome procedure with inherent variability. However, it was recently demonstrated (Margolis-Nunno et al., xe2x80x9cVirus Sterilization in Platelet Concentrates with Psoralen and UVA in the Presence of Quenchersxe2x80x9d Transfusion, 22:541-547 (1992)), that for the inactivation of xe2x89xa76.0 log10 cell-free vesicular stomatitis virus (VSV) by AMT and UVA, the need for oxygen depletion as a means of protecting platelets could be obviated by inclusion of mannitol, a scavenger (quencher) of free radicals. (The addition of quenchers of type I (free radical mediated) or of type II (singlet oxygen mediated) photodynamic reactions is frequently used in other contexts to distinguish which active oxygen species produces a particular photodynamic effect.) Under the conditions used in that study, i.e., 25 xcexcg/ml AMT and 30 minutes of UVA with 2 mM mannitol, the inactivation of cell-free VSV in air was in part oxygen dependent since equivalent virus kill (xe2x89xa76.0 log10) with oxygen depleted required 3 to 4 times more UVA irradiation time (90 minutes to 2 hours).
However, while these methods achieved a high level of kill of cell-free lipid enveloped viruses and of actively replicating, cell-associated virus, non-enveloped viruses and latent cell-associated viruses were not killed to a high extent under the conditions reported therein. Therefore, there was the need to effect the kill of these latter virus forms without causing significant damage to the desired, valuable components in the biological mixture. Conditions which result in the kill of xe2x89xa7106 infectious doses of latent or non-enveloped virus have been shown to modify red blood cells and platelets and result in compromised recovery of labile proteins such as factor VIII.
One of the most successful of the numerous methods developed to inactivate viruses in biological fluids is treatment with organic solvents and detergents; especially treatment with tri(n-butyl)phosphate (TNBP) and non-ionic detergents such as Tween 80 or Triton X-100. See, e.g., U.S. Pat. No. 4,540,573. This method results in excellent recovery of labile proteins, e.g., coagulation factor VIII and IX, while achieving a high level of virus kill, e.g., the killing of xe2x89xa7106 to xe2x89xa7108 ID, of enveloped viruses; however, little inactivation of non-enveloped viruses. See also, U.S. Pat. No. 4,481,189, wherein viral inactivation is by treatment with nonanionic detergent, alcohols, ethers, or mixtures thereof.
Other methods of virus inactivation commonly applied to biological fluids usable in a transfusion setting include treatment with heat at temperatures xe2x89xa760xc2x0 C. or treatment with UVC together with B-propiolactone (B-PL). Each of these methods results either in a significant loss of labile proteins and/or incomplete virus killing. See, e.g., Horowitz, B., Biotechnology of Blood, xe2x80x9cInactivation of viruses found with plasma proteinsxe2x80x9d, Goldstein, J., ed., Butterworth-Heinemann, Stoneham, 417-432, (1991). Additionally, adoption of B-PL has been slow because of its carcinogenicity. Newer methods intended to enhance virus safety are under development. The use of gamma irradiation has been explored in the laboratory, but, thus far, has not been used in the treatment of a commercially available product. See, Horowitz, B., et al., xe2x80x9cInactivation of viruses in labile blood derivatives 1. Disruption of lipid-enveloped viruses by tri(n-butyl)phosphate/detergent combinationsxe2x80x9d, in Transfusion, 25:516-521 (1985); and Singer et al., xe2x80x9cPreliminary Evaluation of Phthalocyanine Photosensitization For Inactivation Of Viral Pathogens in Blood Productsxe2x80x9d, [abstract] British J. Hematology, March 23-25 (1988:Abs. 31). Filters are being developed which appear to remove xe2x89xa7106 ID50 of each of several viruses; however, small viruses, e.g., parvovirus or Hepatitis A virus, would not be expected to be removed completely. Moreover, it is not known whether these filters can be commercially produced with the consistency needed for virus safety.
In spite of these advances, there continues to be a need for novel methods that achieve a high level of kill of both enveloped and non-enveloped viruses without significant loss of labile proteins or other valuable biological components.
The overall objective of the present invention was to achieve a high level of inactivation of both enveloped and non-enveloped viruses in biological compositions without incurring substantial disruption or inactivation of cells meant to be contained therein and without significant loss of labile proteins or other valuable biological components also contained therein. This objective was satisfied with the present invention, which relates generally to a process for inactivating extracellular and intracellular virus in a biological composition without incurring substantial disruption or inactivation thereof, said process comprising subjecting said composition to a virucidally effective amount of irradiation in the presence of (a) a mixture of a compound that quenches type I photodynamic reactions and a compound that quenches type II photodynamic reactions or (b) a bifunctional compound that is capable of quenching both type I and type II reactions, to thereby inactivate said virus while retaining functionality of said composition. The inventive process can, thus, be used to inactivate viruses in whole blood, red blood cell concentrates and platelet concentrates, without adversely affecting red blood cell or platelet structure or function. Similarly, the inventive process can be used to inactivate viruses in biological compositions without incurring substantial inactivation of desired, soluble biological substances (e.g., coagulation factor concentrates, hemoglobin solutions) contained therein.
In accordance with another aspect of the invention, the inventive process is advantageously carried out in the presence of an irradiation sensitizer compound.
In accordance with still another aspect of the invention, the inventive process is advantageously combined with a different virucidal method to enhance virus inactivation.
UV treatment alone of either plasma or AHF concentrates results in a relatively high loss of coagulation factor activity under conditions which kill xe2x89xa7105 ID50 of virus; however, it has been discovered that this loss is significantly reduced (i.e., the recovery is high) when quenchers of photodynamic reactions are added prior to UV treatment. Compare, Murray et al., xe2x80x9cEffect of ultraviolet radiation on the infectivity of icterogenic plasmaxe2x80x9d, in JAMA, 157:8-14 (1955); and, more recently, Kallenbach et al., xe2x80x9cInactivation of viruses by ultraviolet lightxe2x80x9d in Morgenthaler J-J ed. xe2x80x9cVirus inactivation in plasma productsxe2x80x9d, in Cum stud Hematol Blood Transfus., 56:70-82 (1989). Thus, the combined treatment according to the present invention results in a very high level of virus kill while coagulation factor activity is retained at high levels.
Gamma-irradiation of cellular components of blood is the technique of choice for the prevention of transfusion-associated (TA) graft-versus-host (GVHD) as is UVB irradiation of PCs for the prevention of HLA alloimmunization. However, some compromise of RBC (potassium leakage upon storage) and platelet (decreased bleeding time correction) integrity appear to be inherent with current irradiation protocols (Linden, J. V. and Pisciotto, P. 1992, xe2x80x9cTransfusion associated graft-versus-host disease and blood irradiation,xe2x80x9d Trans. Med. Rev., 6:116-123). The inclusion during xcex3-irradiation or UVB irradiation of quenchers (e.g., flavonoids) or quencher mixtures which scavenge both type I and type II photoreaction products will prevent damage to RBCs and platelets under conditions where WBCs are inactivated or otherwise altered. Recent reports of active oxygen species as the major contributors to potassium leakage and red cell membrane damage incurred with xcex3-irradiation (Anderson and Mintz, 1992; Sadrzadeh et al., 1992) support this theory and suggest that the addition of these quenchers will prevent K+ leakage by enhancing the nucleic acid specificity of this WBC inactivation procedure.
In addition, the use of xcex3-irradiation with quencher inclusion as an addition to viral envelope-directed virus sterilization procedures for red blood cell concentrates (RBCCs) and platelet concentrates (PCs) will assure latent virus or provirus inactivation in contaminating lymphocytes.